Schultz-Rogers et al. BMC Medical Genetics
https://doi.org/10.1186/s12881-020-01159-y(2020) 21:219

CASE REPORTOpen Access

Novel loss-of-function variants in TRIO areassociated with neurodevelopmental
disorder: case report
Laura Schultz-Rogers1, Karthik Muthusamy2, Filippo Pinto e Vairo1,2, Eric W. Klee1,2 and Brendan Lanpher2*

Abstract
Background: Damaging variants in TRIO have been associated with moderate to severe neurodevelopmental
disorders in humans. While recent work has delineated the positional effect of missense variation on the resulting
phenotype, the clinical spectrum associated with loss-of-function variation has yet to be fully defined.
Case presentation: We report on two probands with novel loss-of-function variants in TRIO. Patient 1 presentswith a severe neurodevelopmental disorder and macrocephaly. The TRIO variant is inherited from his affectedmother. Patient 2 presents with moderate developmental delays, microcephaly, and cutis aplasia with a frameshift
variant of unknown inheritance.
Conclusions: We describe two patients with neurodevelopmental disorder, macro/microcephaly, and cutis aplasia
in one patient. Both patients have loss-of-function variants, helping to further characterize how these types ofvariants affect the phenotypic spectrum associated with TRIO. We also present the third reported case of autosomal
dominant inheritance of a damaging variant in TRIO.
Keywords: TRIO gene, Autism, Macrocephaly, Microcephaly, Cutis aplasia

Background
The human gene TRIO (triple functional domain) encodes a guanine nucleotide exchange factor (GEF) thatfacilitates the activation of Rho GTPases such as RAC1
[1–3]. Rho GTPases in turn regulate actin cytoskeleton
dynamics and thus play an important role in many neurodevelopmental processes such as migration of neuralprogenitors and neurons during neurogenesis, synapse
formation, and axon outgrowth [4–7]. The TRIO protein
contains several catalytic domains including two GEF
domains. The GEF1 domain activates RAC1 and RHOG
while the GEF2 domain activates RHOA [8–11]. The
protein also contains a c-terminal serine/threonine kinase domain [3]. TRIO is a large gene with 57 exons and* Correspondence: Lanpher.Brendan@mayo.edu
2
Department of Clinical Genomics, Mayo Clinic, Rochester, MN, USA
Full list of author information is available at the end of the article

multiple tissue-specific isoforms, including several different isoforms expressed in the mammalian brain [12–15].
Studies have shown that expression of TRIO peaks inthe brain in the late prenatal period [16] supporting arole for this gene in neurodevelopmental processes. The
importance of this gene in mammalian neurodevelopment has been further highlighted by studies in mice
which have shown complete knockout of the gene to beembryonic lethal, while nervous system-specific knockout mice show abnormal brain morphology, reduced
brain size, and altered learning ability [17–21]. Additionally, it has been shown in vitro that TRIO is importantin regulating synaptic function through regulation of
AMPAr endocytosis at CA1 excitatory synapses [22].
Several patients have been identified with rare or novel
variants in TRIO from large cohort studies examining
whole exome sequencing data from patients with generalized phenotypes such as intellectual disability (ID),© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Schultz-Rogers et al. BMC Medical Genetics(2020) 21:219

neurodevelopmental disorders, schizophrenia, childhood
speech apraxia, autism spectrum disorder (ASD), microcephaly, and epilepsy with intellectual disability [16, 22–35]. Recently, however, several TRIO-specific cohorts
with functional studies have been described that havehelped elucidate the role of pathogenic variation in thisgene in human disease [16, 22, 36–38]. Consistently reported phenotypes described in association with variantsin this gene include global developmental delay, speechdisorder, ID and learning disabilities, and macro- or
microcephaly. Additional phenotypes include behavioral
issues, dysmorphic facies, skeletal/hand anomalies, feeding difficulties, insensitivity to pain, seizures, tremor,
ataxia, abnormal brain MRI, hypotonia, dental anomalies, congenital heart defects, urinary incontinence, andocular defects.
Interestingly, it has become clear that there is a strong
phenotype/genotype correlation specific to missense variants in this gene. Several missense variants that clusterin the spectrin repeat domain have been functionallyshown to lead to increased RAC1 activation and enhanced neurite outgrowth and lamellipodia formation[36] (Fig. 1). Strikingly, all of the patients with missense
variants in this region demonstrated moderate to severe
ID and macrocephaly. Of note, Pengelly et al. also reported a patient with a p.Asn1080Ile missense variant
who displayed severe ID but normal head circumference[37]. Their assessment of this variant in vitro did not
identify an effect on the activation of RAC1, though thisdiscrepancy with previous reports may be due toPage 2 of 6

experimental design. Overall, it is hypothesized that missense variants in this domain cause hyperactivation of
RAC1 leading to the observed macrocephaly phenotype.
This may be due to variants affecting protein folding
and thus interfering with the intermolecular inhibitionof the GEF1 domain by the spectrin repeats [36, 39].
Conversely, missense variants in the RAC1 activating
GEF1 domain have been shown functionally to lead to
reduced overall RAC1 binding and activation, impaired
formation of neurites and lamellipodia, and a reduction
in AMPAR-eEPSC amplitude [16, 36–38] (Fig. 1). The
phenotypes associated with these variants include mild/moderate ID and microcephaly. In contrast to variantsin the spectrin domains, it is hypothesized that variantsin the GEF1 domain lead to loss of normal RAC1 activation and thus the observed microcephaly. The reason forthe severity of ID appears to be greater in association
with spectrin variants is unclear. There has also been
one report of a hypermorphic variant in the GEF2 domain in which functional studies showed increased
RhoA activity [16] (Fig. 1). It is hypothesized that thisdomain is auto-inhibited by the PH2 domain, and variants that disrupt this inhibition lead to hyperactivity.
The phenotype associated with this variant is not welldefined other than schizophrenia.
While clear associations have been shown between thelocation of missense variants and the resulting diseasephenotype, the characterization of framsehift and nonsense variants is less well established. The majority of reported patients with loss-of-function (LOF) variants with

Fig. 1 Variants associated with neurodevelopment disorder in TRIO. Schematic of protein domains of human TRIO (NM_NM_007118.3). Variants
discussed in this report are in black. Variants associated with microcephaly are in blue; microcephaly-associated frameshift/nonsense variants span
the gene, while microcephaly-associated missense variants cluster in the GEF1 domain. Macrocephaly-associated missense variants cluster in theSpectrin repeats domain. With the exception of current patient 1 described here, all frameshift/nonsense variants have to date been associatedwith microcephaly

Schultz-Rogers et al. BMC Medical Genetics(2020) 21:219

occipitofrontal circumference (OFC) data available dodisplay microcephaly. However, the severity of ID andother associated phenotypes does not appear to be consistent. In vitro modeling of truncating variants in the
GEF1 domain has shown reduced RAC1 binding and activation [36–38], while modeling of truncating variantsin the downstream GEF2 domain show show no effecton RAC1 activation. An open question remains as towhether or not these frameshift/nonsense variants are
actually producing a truncated protein in vivo or if thetranscripts are undergoing nonsense-mediated decay(NMD) resulting in haploinsufficiency. The presence ofmultiple alternate transcripts could also result in avariant-specific effect on NMD based on which transcripts are affected. Of the frameshift/nonsense described in TRIO cohorts so far, all would be predicted toundergo NMD based on an in silico prediction tool [40].
However none of the studies mentioned above have actually tested patient samples for absence or presence ofa truncated protein product.
Here we present two patients presenting to the Department of Clinical Genomics at the Mayo Clinic withmoderate/severe developmental delay, facial dysmorphisms, macro- and microcephaly, and a novel phenotype
of cutis aplasia in one patient. Both patients were foundto have presumed LOF variants in TRIO (Fig. 1). Additionally, one of the two probands harbors a variant
inherited from an affected mother, making this the third
documented cases of autosomal dominant (AD) familial
inheritance of disease associated with this gene. The datafrom these patients help expand the known phenotypes
associated with pathogenic TRIO variants, provides further evidence of AD inheritance, and helps elucidate thedisease spectrum associated specifically with presumed
LOF variants.

Case presentation
Clinical description

Patient 1 is a 14-year-old Caucasian boy who was bornto a non-consanguineous union. He presented for genetic evaluation of autism spectrum disorder (ASD). He
was born at term with appropriate birth weight, after an
uncomplicated pregnancy and delivery. He had isolateddelay in attaining socio-adaptive and language milestones and was noted to have repetitive behaviors. He
remains nonverbal and also displays motor stereotypies
such as hand flapping. Significant sleep disturbanceswere noticed since infancy. He was diagnosed with autism spectrum disorder at four years of age. He had febrile seizures during early childhood. He is currently in
middle school with Individualized Education Programand communicates to some extent with augmented communication device. Significant behavioral anger outbursts occurred when he transitioned from elementary

Page 3 of 6

school to middle school which responded to symptomatic medications. There were no self-injurious behaviors
or tactile sensitivities. His family history was significant
for his mother with developmental delay and dyslexia as
well as inflammatory bowel disease, one maternal uncle
with significant developmental disability attributed totraumatic brain injury suffered at age 3, a paternal uncle
with schizophrenia and a maternal 2nd cousin with autism spectrum disorder (Fig. 2b).
On examination the patient was macrocephalic with
OFC of 59 cm (>97th centile). His facial features appeared similar to his mother with broad forehead, deep
set eyes, broad nasal root, prominent columella, andshort philtrum (Fig. 2a). Poor eye contact, low attention
span and motor stereotypies were observed. There wereno neurocutaneous markers and his neurological examination was grossly normal. Standardized psychologicalassessment could not be completed due to limited attention for directed tasks and his gross cognitive testing indicated his functioning at 25-month-old level. His pastevaluation included normal MRI of Brain, EEG andnegative fragile X testing.
Patient 2 is a now 3-year-old boy who presented forgenetic evaluation due to global developmental delay
and congenital anomalies. His parents were nonconsanguineous and of mixed ethnicity (Fig. 2c). Bothparents are generally healthy and had unremarkable developmental histories. His birth weight 2.81 kg (13th percentile), length 49 cm (32nd percentile) and OFC of
32.1 cm (3rd percentile). Cutis aplasia was found after
delivery. He was noted to have a large, irregular skin defect on his posterior scalp with some islands of normal
skin. The calvarium in the affected region was thin.
Three stellate shaped ulcerations involving vertex andocciput of the scalp were noted (largest measuring approximately 4 cm × 3 cm; the next smallest measuring
approximately 3.5 cm × 2.5 cm; and the smallest approximately 1 cm in diameter). The scalp defect was managed
conservatively and healed with scarring and patchy alopecia. At 3 years of age he had predominant language
delay and lagging behind in his head circumference forage. Examination revealed OFC of 46 cm (<3rd percentile). Scalp was notable for extensive scarring and patchy
alopecia. Ears were cupped and mildly simplified. He
had broad forehead, hypertelorism with arched eyebrowsand a slightly upturned nasal tip. Neurological examination was unremarkable except for expressive language
delay. Previous genetic testing included a normal
chromosome microarray analysis.
Genetic analysis

Patient 1 had exome sequencing performed through a
CLIA-certified laboratory. A c.77C > A, p.(Ser26*) pathogenic nonsense variant was reported in TRIO (NM_

Schultz-Rogers et al. BMC Medical Genetics(2020) 21:219

Page 4 of 6

Fig. 2 Characteristics of patients described with novel loss-of-function variants in TRIO. a Photograph of patient 1 displaying macrocephaly, broad
forehead, deep set eyes, broad nasal root, hanging columella and short philtrum. b Pedigree for patient 1. c. Pedigree for patient 2

007118.2). This variant was inherited from the patient’s
affected mother. This variant has not been previously reported in gnomAD, nor has it been previously associatedwith a disease phenotype based on ClinVar or publishedliterature. It is located in exon 1 and would cause truncation in the n-terminal SEC14 domain which lies upstream of the spectrin repeats. In silico prediction of
NMD suggests this variant would cause the affectedtranscript to be targeted for degradation [40]. The function of the SEC14 domain has not been well establishedother than a role in subcellular targeting and possibly
the regulation of endosome dynamics [41]. Patient 1 also
had a c.11G > C, p.(Arg4Pro) missense variant of uncertain significance (VUS) reported in PIK3CA (NM_006218.2). This variant was also maternally inherited.
Due to lack of phenotypic overlap with the associated
MCAP syndrome, CLOVE syndrome, and fibroadipose
hypoplasia, this variant was not thought to be contributing to the patient’s phenotype.
Patient 2 had exome sequencing performed throughthe same clinical laboratory as patient 1. A c.2690dupA,p.(Gln898Alafs*61) pathogenic frameshift variant was reported in TRIO (NM_007118.2). The variant was notinherited from the unaffected mother and a paternal
sample was not available. This variant has not been previously reported in gnomAD, nor has it been previously
reported in ClinVar or published literature. It is locatedin exon 15 and would cause an early truncation in the

spectrin repeats. In silico prediction of NMD also suggest this variant would cause any affected transcript tobe targeted for degradation [40].

Discussion and conclusions
Here we report on two patients with novel pathogenic
presumed LOF variants reported in TRIO. Both patients
have phenotypes overlapping with previously reported
patients including severe (patient 1) and moderate (patient 2) developmental delay, and ID. Both had dysmorphisms including broad/high forehead, deep set eyes,broad nasal root, prominent columella and short philtrum in patient 1 and cupped ears, broad forehead,
hypertelorism, arched eyebrows and a slightly upturned
nasal tip in patient 2. Of note, Ba et al. also reported several patients with high forehead in their cohort [22]. A
novel phenotype presenting in patient 2 includes extensive cutis aplasia which has not been previously reported
and may represent an expanded phenotype associatedwith this disorder.
Microcephaly was reported in patient 2 which matches
well with most previously characterized patients with
nonsense/truncating variants (including p.(Arg217*),p.(Gln768*),p.(Asp1251Valfs*11),p.(Trp1376*),p.(Gln1489Argfs*11), p.(Arg1620Serfs*10), p.(Val1698Leufs*61),p.(Lys1918*),p.(Leu2031Phefs*9),andp.(Phe2472Serfs*54)) [16, 22, 35–37]. Notably, patient 2
does not show a severe ID phenotype or macrocephaly

Schultz-Rogers et al. BMC Medical Genetics(2020) 21:219

as is reported for patients with missense variants in thesame spectrin repeat domain, reinforcing that microcephaly is associated with the majority of presumed LOF
variants and thereby with haploinsufficiency of the gene.
Interestingly, patient 1 had macrocephaly in contrast
to the majority of other patients with truncating andpresumed LOF variants. Barbosa et al. described a groupof non-related individuals with macrocephaly who had
missense variants clustering on the spectrin repeat domain. The authors performed in vitro experiments andshowed that the missense variants at the spectrin repeat
7 cause a gain-of-function effect, inducing hyperactivation of RAC1. None of the individuals with presumable
LOF variants in their cohort presented with macrocephaly [36]. One possibility as to why patient 1 seems tohave a different phenotype may be that the variant is in
the far N-terminal region and therefore there are severaltranscripts with downstream alternate start sites that
couldbeunaffectedbythisvariant(ENST00000509967.2,
ENST00000513206.1,
ENST00000344135.5). Variations in the location andtiming of expression of these alternate transcripts may
explain the discrepancy in observed phenotypes between
patient 1 and other patients with presumed LOF
variants.
Overall, we demonstrate two cases with patients presenting with neurological phenotypes and reported
pathogenic, presumed LOF variants in TRIO. We add to
the known clinical phenotype spectrum associated withthis gene with the report of cutis aplasia in patient 2.
We also add to the literature the third documented caseof familial autosomal dominant inheritance of a TRIOassociated disorder highlighting the variable expressivity
associated with TRIO disorders. Moreover, we report ona patient with macrocephaly and a nonsense variant upstream to the domain hotspot that has been associatedwith this phenotype. Future work clarifying the status oftranscript degradation/protein translation for truncating
and nonsense variants in patient samples, especially testing for hyperactivation of RAC1 in the individual with anonsense variant is needed to fully characterize the effect of these types of variants.
Abbreviations
TRIO: Triple functional domain; GEF: Guanine nucleotide exchange factor;
ID: Intellectual disability; ASD: Autism spectrum disorder; OFC: Occipitofrontal
circumference; NMD: Nonsense-mediated decay; LOF: Loss-of-function;
AD: Autosomal dominant; HGVS: Human Genome Variation Society

Acknowledgements
We would like to thank the families for participating in this research.

Consent to participate
Parents of all of the participants provided written consent to participate in
the study.

Page 5 of 6

Authors’ contributions
All authors read and approved the manuscript. LSR performed variant
analysis and wrote the manuscript. KM performed clinical examination andcare of the patients, contributed to writing, intellectual content and revisionof the manuscript. FPV assisted in variant analysis and editing of themanuscript. EWK supported this research and contributed to writing andediting of the manuscript. BL provided clinical examination and care of thepatients, contributed to writing, intellectual content and critical revision of
the manuscript. All the authors approved the submitted version and themodified version.
Funding
This work was supported by the Mayo Clinic Center for Individualized
Medicine (CIM), the Investigative and Functional Genomics Program and theWilliam O. Lund, Jr. and Natalie C. Lund Charitable Foundation.
Availability of data and materials
Genomic sequence data cannot be deposited due to restrictions imposed by
the IRB. The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly available
due to their containing information that could compromise the privacy of
research participants.
Ethics approval and consent to participate
This study was approved by the Mayo Clinic institutional review board. IRB#:
12–009346. Written consent was obtained from the parents or legal
guardians of all participants since the patients were under the age of 16.
Consent for publication
Written informed consent for publication of identifying images and otherpersonal or clinical details was obtained from the parents or legal guardianssince the participants were under the age of 18. A copy of the writtenconsent is available for review by the Editor of this journal.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA.
2
Department of Clinical Genomics, Mayo Clinic, Rochester, MN, USA.
Received: 23 July 2020 Accepted: 28 October 2020

References
1. Schmidt S, Debant A. Function and regulation of the rho guanine
nucleotide exchange factor trio. Small GTPases. 2014;5:e29769.
2. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;
420(6916):629–35.
3. Debant A, Serra-Pages C, Seipel K, O'Brien S, Tang M, Park SH, Streuli M. The
multidomain protein trio binds the LAR transmembrane tyrosine
phosphatase, contains a protein kinase domain, and has separate racspecific and rho-specific guanine nucleotide exchange factor domains. Proc
Natl Acad Sci U S A. 1996;93(11):5466–71.
4. Govek EE, Newey SE, Van Aelst L. The role of the rho GTPases in neuronaldevelopment. Genes Dev. 2005;19(1):1–49.
5. Cannet A, Schmidt S, Delaval B, Debant A. Identification of a mitotic RacGEF, trio, that counteracts MgcRacGAP function during cytokinesis. Mol Biol
Cell. 2014;25(25):4063–71.
6. Ba W, van der Raadt J, Nadif Kasri N. Rho GTPase signaling at the synapse:
implications for intellectual disability. Exp Cell Res. 2013;319(15):2368–74.
7. Herring BE, Nicoll RA. Kalirin and trio proteins serve critical roles in
excitatory synaptic transmission and LTP. Proc Natl Acad Sci U S A. 2016;
113(8):2264–9.
8. Bellanger JM, Lazaro JB, Diriong S, Fernandez A, Lamb N, Debant A. The two
guanine nucleotide exchange factor domains of trio link the Rac1 and the
RhoA pathways in vivo. Oncogene. 1998;16(2):147–52.
9. Blangy A, Vignal E, Schmidt S, Debant A, Gauthier-Rouviere C, Fort P.
TrioGEF1 controls Rac- and Cdc42-dependent cell structures through thedirect activation of rhoG. J Cell Sci. 2000;113(Pt 4):729–39.

Schultz-Rogers et al. BMC Medical Genetics(2020) 21:219

10. Chhatriwala MK, Betts L, Worthylake DK, Sondek J. The DH and PH domainsof trio coordinately engage rho GTPases for their efficient activation. J Mol
Biol. 2007;368(5):1307–20.
11. Jaiswal M, Dvorsky R, Ahmadian MR. Deciphering the molecular andfunctional basis of Dbl family proteins: a novel systematic approach towardclassification of selective activation of the rho family proteins. J Biol Chem.
2013;288(6):4486–500.
12. McPherson CE, Eipper BA, Mains RE. Multiple novel isoforms of trio are
expressed in the developing rat brain. Gene. 2005;347(1):125–35.
13. Portales-Casamar E, Briancon-Marjollet A, Fromont S, Triboulet R, Debant A.
Identification of novel neuronal isoforms of the rho-GEF trio. Biol Cell. 2006;
98(3):183–93.
14. Yoshizuka N, Moriuchi R, Mori T, Yamada K, Hasegawa S, Maeda T, Shimada
T, Yamada Y, Kamihira S, Tomonaga M, et al. An alternative transcriptderived from the trio locus encodes a guanosine nucleotide exchange
factor with mouse cell-transforming potential. J Biol Chem. 2004;279(42):43998–4004.
15. Ma XM, Huang JP, Eipper BA, Mains RE. Expression of trio, a member of the
Dbl family of rho GEFs in the developing rat brain. J Comp Neurol. 2005;
482(4):333–48.
16. Katrancha SM, Wu Y, Zhu M, Eipper BA, Koleske AJ, Mains RE.
Neurodevelopmental disease-associated de novo mutations and rare
sequence variants affect TRIO GDP/GTP exchange factor activity. Hum Mol
Genet. 2017;26(23):4728–40.
17. Peng YJ, He WQ, Tang J, Tao T, Chen C, Gao YQ, Zhang WC, He XY, Dai YY,
Zhu NC, et al. Trio is a key guanine nucleotide exchange factor
coordinating regulation of the migration and morphogenesis of granulecells in the developing cerebellum. J Biol Chem. 2010;285(32):24834–44.
18. Zong W, Liu S, Wang X, Zhang J, Zhang T, Liu Z, Wang D, Zhang A, Zhu M,
Gao J. Trio gene is required for mouse learning ability. Brain Res. 1608;2015:
82–90.
19. O'Brien SP, Seipel K, Medley QG, Bronson R, Segal R, Streuli M. Skeletal
muscle deformity and neuronal disorder in trio exchange factor-deficientmouse embryos. Proc Natl Acad Sci U S A. 2000;97(22):12074–8.
20. Katrancha SM, Shaw JE, Zhao AY, Myers SA, Cocco AR, Jeng AT, Zhu M,
Pittenger C, Greer CA, Carr SA, et al. Trio Haploinsufficiency causes
neurodevelopmental disease-associated deficits. Cell Rep. 2019;26(10):2805–
17 e2809.
21. Tao T, Sun J, Peng Y, Wang P, Chen X, Zhao W, Li Y, Wei L, Wang W, Zheng
Y, et al. Distinct functions of trio GEF domains in axon outgrowth of
cerebellar granule neurons. J Genet Genomics. 2019;46(2):87–96.
22. Ba W, Yan Y, Reijnders MR, Schuurs-Hoeijmakers JH, Feenstra I, Bongers EM,
Bosch DG, De Leeuw N, Pfundt R, Gilissen C, et al. TRIO loss of function isassociated with mild intellectual disability and affects dendritic branchingand synapse function. Hum Mol Genet. 2016;25(5):892–902.
23. Carraro M, Monzon AM, Chiricosta L, Reggiani F, Aspromonte MC, Bellini M,
Pagel K, Jiang Y, Radivojac P, Kundu K, et al. Assessment of patient clinicaldescriptions and pathogenic variants from gene panel sequences in the
CAGI-5 intellectual disability challenge. Hum Mutat. 2019;40(9):1330–45.
24. Geisheker MR, Heymann G, Wang T, Coe BP, Turner TN, Stessman HAF,
Hoekzema K, Kvarnung M, Shaw M, Friend K, et al. Hotspots of missense
mutation identify neurodevelopmental disorder genes and functional
domains. Nat Neurosci. 2017;20(8):1043–51.
25. Kosmicki JA, Samocha KE, Howrigan DP, Sanders SJ, Slowikowski K, Lek M,
Karczewski KJ, Cutler DJ, Devlin B, Roeder K, et al. Refining the role of denovo protein-truncating variants in neurodevelopmental disorders by using
population reference samples. Nat Genet. 2017;49(4):504–10.
26. Deciphering Developmental Disorders S. Prevalence and architecture of denovo mutations in developmental disorders. Nature. 2017;542(7642):433–8.
27. Iossifov I, O'Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, Stessman
HA, Witherspoon KT, Vives L, Patterson KE, et al. The contribution of denovo coding mutations to autism spectrum disorder. Nature. 2014;
515(7526):216–21.
28. Jansen S, Hoischen A, Coe BP, Carvill GL, Van Esch H, Bosch DGM, Andersen
UA, Baker C, Bauters M, Bernier RA, et al. A genotype-first approach
identifies an intellectual disability-overweight syndrome caused by PHIP
haploinsufficiency. Eur J Hum Genet. 2018;26(1):54–63.
29. Eising E, Carrion-Castillo A, Vino A, Strand EA, Jakielski KJ, Scerri TS,
Hildebrand MS, Webster R, Ma A, Mazoyer B, et al. A set of regulatory genes
co-expressed in embryonic human brain is implicated in disrupted speech
development. Mol Psychiatry. 2019;24(7):1065–78.

Page 6 of 6

30. Wang T, Guo H, Xiong B, Stessman HA, Wu H, Coe BP, Turner TN, Liu Y,
Zhao W, Hoekzema K, et al. De novo genic mutations among a Chinese
autism spectrum disorder cohort. Nat Commun. 2016;7:13316.
31. de Ligt J, Willemsen MH, van Bon BW, Kleefstra T, Yntema HG, Kroes T,
Vulto-van Silfhout AT, Koolen DA, de Vries P, Gilissen C, et al. Diagnostic
exome sequencing in persons with severe intellectual disability. N Engl J
Med. 2012;367(20):1921–9.
32. Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ,
Ercan-Sencicek AG, DiLullo NM, Parikshak NN, Stein JL, et al. De novo
mutations revealed by whole-exome sequencing are strongly associatedwith autism. Nature. 2012;485(7397):237–41.
33. Lionel AC, Costain G, Monfared N, Walker S, Reuter MS, Hosseini SM,
Thiruvahindrapuram B, Merico D, Jobling R, Nalpathamkalam T, et al.
Improved diagnostic yield compared with targeted gene sequencing panels
suggests a role for whole-genome sequencing as a first-tier genetic test.
Genet Med. 2018;20(4):435–43.
34. Boonsawat P, Joset P, Steindl K, Oneda B, Gogoll L, Azzarello-Burri S, ShethF, Datar C, Verma IC, Puri RD, et al. Elucidation of the phenotypic spectrum
and genetic landscape in primary and secondary microcephaly. Genet Med.
2019;21(9):2043–58.
35. Kahrizi K, Hu H, Hosseini M, Kalscheuer VM, Fattahi Z, Beheshtian M, Suckow
V, Mohseni M, Lipkowitz B, Mehvari S, et al. Effect of inbreeding on
intellectual disability revisited by trio sequencing. Clin Genet. 2019;95(1):151–9.
36. Barbosa S, Greville-Heygate S, Bonnet M, Godwin A, Fagotto-Kaufmann C,
Kajava AV, Laouteouet D, Mawby R, Wai HA, Dingemans AJM, et al.
Opposite modulation of RAC1 by mutations in TRIO is associated with
distinct, domain-specific neurodevelopmental disorders. Am J Hum Genet.
2020;106(3):338–55.
37. Pengelly RJ, Greville-Heygate S, Schmidt S, Seaby EG, Jabalameli MR, Mehta
SG, Parker MJ, Goudie D, Fagotto-Kaufmann C, Mercer C, et al. Mutations
specific to the Rac-GEF domain of TRIO cause intellectual disability andmicrocephaly. J Med Genet. 2016;53(11):735–42.
38. Sadybekov A, Tian C, Arnesano C, Katritch V, Herring BE. An autism
spectrum disorder-related de novo mutation hotspot discovered in the
GEF1 domain of trio. Nat Commun. 2017;8(1):601.
39. Chen SY, Huang PH, Cheng HJ. Disrupted-in-schizophrenia 1-mediated axon
guidance involves TRIO-RAC-PAK small GTPase pathway signaling. Proc Natl
Acad Sci U S A. 2011;108(14):5861–6.
40. Coban-Akdemir Z, White JJ, Song X, Jhangiani SN, Fatih JM, Gambin T,
Bayram Y, Chinn IK, Karaca E, Punetha J, et al. Identifying genes whosemutant transcripts cause dominant disease traits by potential gain-offunction alleles. Am J Hum Genet. 2018;103(2):171–87.
41. Saito K, Tautz L, Mustelin T. The lipid-binding SEC14 domain. Biochim
Biophys Acta. 2007;1771(6):719–26.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.